Cloning of protein A DNA to OmpA constructs

1. Isolation of plasmids from cultures inocluated on previous day (pACYC177+OmpA_A_omega).
2. Control digest of isolated plasmids with BamHI and SacI.

Cloning of protein Z DNA to pET15b-OmpA-omega in place of OmpA
Paweł

1. Results of ligation: 6 colonies grown.
2. Each colony cultured overnight in LB + ampicillin

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin:

1. Isolation of plasmids from transformants
2. Digest of plasmids with SacI and NotI
3. Gel electrophoresis of products
4. 2 selected clones were send to DNA sequencing lab

Cloning of protein Z DNA to pET15b-OmpA-omega in place of OmpA
Paweł

1. Isolation of plasmids from cultures inocluated on previous day
2. Control digest of isolated plasmids with NdeI and NotI. Zero positive results obtained - just empty vectors
3. Another overnight ligation of pET15b-OmpA-omega (NdeI/NotI cutted) with protein Z DNA

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin:

1. Since the phage is gone we can continue this cloning:
2. Restriction digest of PCL product and pKS vector with SacI and NotI
3. Ligation of omega-A fusion with pKS vector
4. Transformation of Top10 with ligation product

Cloning of protein A DNA to OmpA constructs

1. Digest of pKS-A plasmid, pACYC177+OmpA_omega and pACYC177+OmpA_alpha with NotI and SacI (pACYC177 were also dephosporylated with CIAP)
2. Gel electrophoresis and gel-out of proper bands (420 bp - pKS-A lane, 4050 bp - pACYC177+OmpA_omega lane and 4300 bp pACYC177+OmpA_alpha lane).
3. Ligation of A DNA fragment with both pACYC177 vectors.
4. Transformation of E. coli TOP10 strain with ligations.
5. Transformants plating on LB + kanamycin.

Cloning of protein Z DNA to OmpA constructs
Michał K.

1. Isolation of plasmids from cultures inocluated on previous day (pACYC177+OmpA_Z_alpha).
2. Control digest of isolated plasmids with BamHI and SacI (we found 4 good clones).

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